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The Evolution Of Mutation Detections Techniques Biology Essay

Published: 23, March 2015

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The evolution of mutation detection techniques has been influenced by several factors e.g. cost-effectiveness, accuracy and high-throughput status (Gross et. al., 1999), techniques such as Direct Sequencing which has a clear evolution leading to the era of "next generation DNA sequencing" (e.g. Applied Biosystems ABI SOLiD system) (Ansorge et. al., 2009). Direct sequencing has also provided the foundation for the development of other mutation detection techniques such as Fluorescent fingerprinting which is based on fluorescent Sanger sequencing (a variation of Direct Sequencing) (Liu et. al., 1998).

Results from these techniques have to be accurate and available fast as mutation screening allows the identification of at risk individuals in families with a history of the specific disease translating into better patient management. Mutation screening is important as many diseases do not exhibit early warning signs e.g. Hypertrophic cardiomyopathy in which the first symptom is (in most cases) sudden cardiac death (Maron et al., 1982). In debilitating diseases, e.g. Parkinson's disease (Chaudhuri et. al., 2006), an individual's knowledge of his/her mutation status may affect his/her choice to have children.

Whether your interest is clinical - or research-based, mutation screening is a powerful tool in understanding pathogenesis and improving treatment.

There is a growing trend in detection technique development of a reduction in waste and the use of radioactive reagents - which are toxic to both the user and environment. This is the focus of the Massachusetts' Institute of Technology's (MIT) awareness program (http://web.mit.edu/environment/academic/green_chemicals.html). The MIT awareness program is based on the concept of Green Chemistry (an approach to designing, manufacturing and usage of reagents to reduce or eliminate chemical hazards (Anastas et. al., 1998).

This literature review will explore the evolution of mutation techniques within themselves and how they relate to one another.

Direct Sequencing

Direct sequencing using DNA polymerase and 2', 3'-dideoxynucleotides was first described in 1977 by Sanger and colleagues (Sanger et. al., 1977). DNA polymerase adds nucleotides to the 3' end of the template and terminators (dideoxynucleotides) terminate nucleotide addition to the template due to the absence of a 3' hydroxyl group on the terminators (Fig. 1) (Sanger et. al., 1977).

The process of nucleotide addition commences when a primer, a template, DNA polymerase and terminators are incubated together in the presence of dideoxy thymine triphosphate (ddTTP) and deoxynucleotide triphosphates (dNTPs) - one of which is 32P-labelled (radioactively-labelled) (Sanger et. al., 1977). The resultant fragments are electrophoresed on a denaturing acrylamide gel and the DNA sequence can be read off from the banding pattern after the gel is subjected to autoradiography (Sanger et. al., 1977). Direct sequencing has evolved and is still used today and is the validation method of choice although these days the process is automated and is more rapid (Smith et. al., 1987). Automated Sanger sequencing involves fluorescently-labelling the ddNTPs (Fig. 2) (Smith et. al., 1987). The fluorescently-labelled ddNTPs passes the DNA laser in the DNA Sequencer, exciting the fluorophores resulting in a fluorescent signal from which the nucleotide can be identified as a different fluorophores is used for each nucleotide (Smith et. al., 1987).

Denaturing Gradient Gel Electrophoresis

In 1979 Fischer and colleagues noted that an ascending denaturant gradient, i.e. 40% - 75% denaturant [100 % denaturant is equal to 7M urea/40% (vol/vol) Formamide], has the ability to separate fragments of various length according to their structure (Fischer et. al., 1979). The structure of the fragments is altered in the case of base-pair changes, deletions and insertions, this structure change would affect the electrophoretic mobility (the migratory pattern of the sample through the gel) (Fischer et. al., 1979). Fischer and co-workers utilised two-dimensional gel electrophoresis, whereby a SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis (PAGE) takes place in one direction and then at right angles to the previous direction (http://genome.wellcome.ac.uk/doc_WTD021045.html) (Fischer et. al., 1979). The first dimension of the gel is subjected to isoelectric-focusing (the sample is loaded in the middle of the left portion of the gel, where the pH is neutral, and a voltage is applied across the gel). Migration of the sample takes place until the sample has reached its isoelectric point (where the charge of the molecule is equal to the charge of its immediate environment) (Fischer et. al., 1979). The second dimension of the gel is subjected to SDS-PAGE to separate the fragments. The fragments which were subjected to restriction endonuclease treatment prior to polyacrylamide gel electrophoresis (at 60Â°C) stop migrating when they reach a certain denaturant concentration which is determined by the sequence of the molecule. Sheffield and colleagues attached a GC-clamp (40-45 base pairs) to either end of the genomic or cloned DNA fragments by PCR amplification to increase detection sensitivity of single-base changes (Fig. 3), as a GC-clamp prevents complete denaturation of the amplicons (http://bccm.belspo.be/newsletter/17-05/bccm02.htm) (Sheffield et. al., 1989). According to the study DGGE is also extremely reliable in heterozygosity detection (screening for the presence of two different alleles of the same gene in a sample) (Sheffield et. al., 1989). In a study done by Wijnen and colleagues it was revealed that DGGE-based mutation detection could not detect large genomic deletions of promoter mutations, but they found the approach to be economical as well as a potentially important pathogenic mutation detection method in Hereditary Nonpolyposis Colorectal Cancer (HNPCC) (Wijnen et. al., 1996).

Pyrosequencing

Nyrén and colleagues developed pyrosequencing on the principle than during DNA synthesis inorganic pyrophosphate (PPi) is released (Nyrén et. al., 1987).

Figure 4: Pyrosequencing. Extracted from Fakhrai-Rad et. al. 2002

Sequential Protocol

Cyclic ProtocolPyrosequencing utilises an enzymatic cascade (Fig. 4) in which DNA polymerase adds the dNTP to the nucleic acid chain releasing PPi which is converted to ATP by ATP Sulfurylase (Nyrén et. al., 1987; Fakhrai-Rad et. al., 2002). To prevent false positives Apyrase is added to degrade dNTPs which haven't been added and any residual AMP from the ATP Sulfurylase reaction. Luciferase subsequently converts the ATP to light - via luciferin oxidation - which is detected by a Luminometer (Photon detector) (Nyrén et. al., 1987). The Luminometer results are used to detect the DNA sequence and allelic frequency of specific nucleotides as luminometer results are visualized as peaks (Nyrén et. al., 1987). When a nucleotide is added to one of the alleles a 0.5 peak forms, if a nucleotide is added to both alleles a 1.0 peak forms (Fig. 5) also peak formation can be resultant of consecutive identical nucleotide additions and the specific nucleotide i.e. A, T, C or G peaks are visualised in different colours to distinguish between them (Ahmadian et. al., 2000). In all cases the first nucleotide is the same within the sample group to normalise the peak values and to act as an internal marker (Ahmadian et. al., 2000). There are two nucleotide addition protocols available i.e. Cyclic and Sequential (Fig. 5) (Ahmadian et. al., 2000).

Sequential Protocol

Cyclic Protocol

Sequence for the SNP

Figure 5: Sequencing protocols. Adapted from Ahmadian et. al. 1999

Either cyclic or sequential protocol could be use as both are reliable but the sequential protocol is faster than the cyclic protocol but has the disadvantage of not generating a comprehensive pattern difference as is illustrated in Fig. 6 (Ahmadian et. al., 2000). Pyrosequencing is ideal for large-scale studies as it enables rapid real-time sequence determination of 20-30 base pairs (Ahmadian et. al., 2000). It can be automated and is extremely accurate and easy as it produces specific patterns for specific SNP variant allele combinations (Ahmadian et. al. 1999). As a major shortcoming this technique is its time-consuming template preparation (Fig. 6) Nordstrom and co-workers proposed the substitution of ssDNA with dsDNA (Nordstrom et. al. 2000). As apyrase cannot degrade PPi released during PCR (PCR-PPi) great care has to be taken to ensure that effective PCR purification takes place - the removal of residual primers, nucleotides and especially PPi - as any leftover components would interfere with sequencing. Reassociation of template strands would also compromise primer-template hybridisation. In this study the data obtained using dsDNA as a template was of a similar standard as in the case of ssDNA template utilisation (Fig. 7) (Nordstrom et. al., 2000). Applications of this technique are SNP genotyping, SNP discovery, Haplotyping and Allelic frequency studies (Fakhrai-Rad H. et. al. 2002; Ahmadian et. al. 1999).

Ligation-mediated gene detection technique

The Ligation-mediated gene detection technique was devised in 1988 by Landegren and colleagues (Landegren et. al., 1988). Their strategy permitted the distinction between known sequence variants which differ by at least a single base.

Figure 8: Ligation-mediated gene detection technique.

Extracted from Landegren et. al., 1988

Ligation-mediated gene detection utilises oligonucleotide probes which are allowed to hybridise to denatured test DNA, the oligonucleotide probes are then ligated - provided they juxtapose and the oligonucleotides are correctly base-paired at the region of ligation (Fig. 8) (Landegren et. al., 1988). One of the oligonucleotide probes is biotin-labelled (indicated with a B in Fig. 8) and the other is 32P-labelled (indicated with an asterisk in Fig. 8), resulting in the biotinylated oligonucleotide probes being bound to the streptavidin (which is immobilised to a solid support) after ligation (Landegren et. al., 1988). Biotin-labelled oligonucleotide probes that are ligated to radioactively-labelled oligonucleotide probes would be detected when the solid support is subjected to autoradiography (Landegren et. al., 1988). This strategy could be set up to analyse multiple loci by adding a nonhybridizing 3' sequence expansion unique in length to each unlabelled oligonucleotide per set of allele-specific oligonucleotides (Landegren et. al., 1988). Nickerson DA et. al. (1990) described how the advancement of Ligation-mediated detection, the oligonucleotide ligation assay (OLA) could be automated (Fig 9) by performing an Enzyme-linked Immunosorbent Assay (ELISA) (Fig. 10) with a robotic workstation; in addition this method makes use of a nonisotopic reporter probe, i.e. Digoxigenin, instead of a radioactively-labelled probe (tying in the principle of green chemistry). Other advantages of this method are that the reagents are stable and the entire assay is performed in microtiter wells as it makes use of ELISA which eliminates the need for centrifugation and electrophoresis (Nickerson et. al. 1990). It is a powerful approach to high-resolution genetic linkage mapping as well as human leukocyte antigen (HLA) typing, oncogene analysis, and infectious pathogen identification (Nickerson et. al. 1990).

In the study a nucleotide was added to the 3' end of 30-mer and 20-mer oligonucleotides, both are complementary to the same genomic sequence, to determine the adjacent nucleotides- each indicated with an asterisk in Fig. 11 (Sokolov, 1989). Ghosh and colleagues improved on the primer extension technique by making allowing it to scan multiple samples simultaneously i.e. multiplex, also the newer technique utilises fluorescence (Fig. 12) instead of radioactive reagents to detect mutations (Ghosh et. al., 1996). Other improvements on the original primer extension technique are that heteroduplex formation during PCR amplification does not hinder detection, it is less labour-intensive and time-consuming than the original primer extension technique (Ghosh et. al., 1996) as both the wild-type and mutant DNA can be probed in the same reaction. The only disadvantage of the technique is that the required equipment is expensive (Ghosh et. al., 1996). Fluorescence-based primer extension's application in mitochondrial and Alzheimer's disease mutation detection was studied by Fahy and colleagues (Fahy et. al., 1997).

Heteroduplex analysis

Heteroduplex analysis originated from the occurrence of heteroduplex formation (molecules which are recombinants of mutant and wild type molecules) of amplified PCR products of homologous loci during a study on mYfin, the mouse testis-determining Y gene (Nagamine et. al. 1989). The heteroduplex analysis (Fig. 13) relies on the fact that single-stranded DNA which has genetically recombined to form a double-stranded DNA structure would have a different gel migratory pattern than one that hasn't recombined (Nagamine et. al. 1989). In a study on autosomal dominant retinitis pigmentosa (ADRP) Keen and colleagues utilised denatured, undigested 211bp exon-encompassing fragments to perform HET for the detection of single base pair mismatches (Keen et. al., 1991). Its general reliability couldn't be proved as the study was utilised to detect only the four known mutations at the time of the study (Keen et. al., 1991) - there are over 40 mutations found to be ARDP-causing (Sung, C.H. et. al., 1993) presently. In their opinion this technique was simpler than RNase digestion, chemical cleavage, GC clamping (DGGE), Single-Strand Conformation Polymorphism (SSCP), as well as techniques making use of a temperature gradient (Keen et. al. 1991).

More recently it has been discovered by Vallian and Nasiri that sodium bisulphite treatment - which chemically converts cytosine residues to uracil residues in DNA (Hayatsu H. 1976 ) would also convert complementary DNA strands to non-complementary DNA strands, reducing the risk of reannealing - this is important as reannealing interferes with band resolution (Vallian and Nasiri, 2010). This technique is known as Deaminated SSCP (DSSCP) (Fig. 15) and provides an easy and high resolution analysis of single nucleotide changes in a DNA fragment, making it more high-throughput than its predecessor (Vallian and Nasiri, 2010).

Dideoxy fingerprinting

Dideoxy fingerprinting (ddF) was first described in a study on the human factor IX gene by Sarkar and colleagues in 1992 (Sarkar et. al., 1992). It can detect all single-nucleotide alterations in the gene, it provides the location of the sequence change and detection efficiency is independent on the amplicon length (Sarkar et. al., 1992). Dideoxy fingerprinting (Fig. 16) utilises the four standard dideoxy sequencing reactions (Fig. 17) to generate a banding pattern which can be visualised by electrophoresis on a non-denaturing polyacrylamide gel from which mutations are detected as either a gain or loss of a dideoxy termination segment or by a change in electrophoretic mobility (Liu Q et. al., 1996). Dideoxy fingerprinting is rapid and has a low frequency of false positives (Sarkar et. al. 1992). Also required amplification is reduced as a large DNA segment can be amplified in one PCR reaction and then screened in smaller segments (Sarkar et. al., 1992). The technique was modified by Liu and colleagues to enable it to perform a customized cycle sequencing protocol - Sanger reactions are performed in both the upstream and downstream directions simultaneously - this technique is known as Bi-directional ddF (Bi-ddF) (Liu Q et. al., 1996). Bi-directional ddF (Fig. 18) was utilised to perform an analysis on the human factor IX gene and produced reliable mutation detection results of a larger region than SSCP, as a mutation found in large suboptimal resolved segments in one direction can be analysed in smaller optimally resolved segments in the opposite direction (Liu et. al., 1996).

Denaturing High-Performance Liquid Chromatography

Denaturing high-performance liquid chromatography (DHPLC) was developed in 1995 by Oefner and Underhill (Oefner and Underhill, 1995). Its basic principle (Fig. 19) is that heteroduplexes which are formed by renaturation of wild type and mutant alleles of the same gene would elute differently than homoduplexes (molecules which have alleles for either the wild type or mutant genotype) during liquid chromatography under partially denaturing conditions (Oefner and Underhill, 1995).

DHPLC allows single-base substitutions in addition to small insertions and deletions to be detected efficiently (Underhill et. al., 1996). The reliability and accuracy of DHPLC for detection of somatic and germline mutations was proved by Liu and colleagues in their study on RET (Rearranged transforming proto-oncogene), PTEN (Phosphate and tensin homologue deleted on chromosome ten gene) and CFTR (Cystic Fibrosis transmembrane conductance regulator gene), except in cases where GC-content was exceptionally high (RET exon 10=64% and PTEN exon 3= 27%) (Liu et. al., 1998). This study confirmed that it is a reliable method to create a chromatogram based on sample heterogeneity. Jones and colleagues compared DHPLC to DGGE and found DHPLC to be less laborious and more cost-effective (Jones et. al., 1999). Also DHPLC is better suited to screen large sample groups due to the small amount of handling required and its high-throughput status than DGGE, but the technique has to be optimized to efficiently screen for TP53 mutations (has been linked to higher risk of malignancies and early cancer development) in oesophageal and cardiac cancers (Breton J et. al., 2003). Moreover since DHPLC can be automated, the investigators rightly concluded that DHPLC was superior to DGGE (Jones et. al., 1999). A study by Gross and colleagues (1999) on the BRCA1 gene confirmed that DHPLC was less time-consuming than SSCP and Direct Sequencing (Gross et. al., 1999). Furthermore, the addition of a fraction collector to the liquid chromatography equipment allows for the collection of injected samples which could be used in further applications such as direct sequencing for validation of results - as direct sequencing requires highly purified DNA samples (Gross et. al., 1999). According to a study by Young and colleagues on Hereditary Non-Polyposis Colon Cancer (HNPCC) - specifically tumour suppressor genes - DHPLC can detect deletions, insertions and base substitutions (Young et. al., 2002).

DHPLC has since evolved with the invention of the condition-orientated-PCR primer-embedded reactor (COPPER) plate by Kosaki and colleagues (Kosaki et. al., 2005) and was validated by Yuan and colleagues for use in HNPCC mutation detection specifically hMLH1 and hMSH2 genes (Yuan et. al., 2006). This is a vital technique in the study of hereditary nonpolyposis colorectal cancer (HNPCC) and its hMLH1 and hMSH2 genes (Yuan et. al., 2006). COPPER-plate technology reduces the time required to prepare samples and technical skill, it also standardizes mutation screening of the two genes (Yuan et. al., 2006). DHPLC coupled with COPPER plate technology has proven to be more cost-effective and rapid than DHPLC alone, as the COPPER plate-DHPLC can amplify all 35 exons -both genes in total - in one PCR reaction, this was previously impossible due to their different Tm (melting temperature) (Yuan et. al., 2006). Preparation time is decreased as one mastermix cocktail is required for the amplification of both the genes (Yuan et. al., 2006). If wild type (WT) genomic DNA is added to the mastermix all the mutation possibilities can be screened for. All the results were confirmed by dye-terminator sequencing, and revealed no false positives. The COPPER plate-DHPLC technique could detect SNPs as well as deletions, insertions and repeat polymorphisms and accurate analysis of fragments up to 600bp could be performed (Yuan et. al., 2006).

Fluorescent Fingerprinting

Fluorescent fingerprinting (FF) was developed by Liu and colleagues in 1998 (Liu et. al., 1998). It utilises fluorescently-labelled dideoxy nucleotide triphosphates (F-ddNTPs) to label PCR amplicons during the Sanger's chain termination reaction (see Fig. 2) which uses a fluorophore for each nucleotide (Liu et. al., 1998). PCR Amplicons are electrophoresed using a ABI-373 Sequencer, which generates a pattern based on the fluorescently-labelled nucleotide which passes through the laser scanning window (Liu et. al., 1998). This is converted into a colour-nucleotide associated fingerprint. In their study Liu and co-workers compared the fingerprints of several samples to determine whether a sequence change was present (a difference in the fingerprint was considered to be caused by a sequence variant) (Liu et. al., 1998). Mutation-positive samples were sequenced and compared to the wild-type sequence to determine the base-substitution, microdeletion or microinsertion. The FF technique has been successfully used in the identification of Haemophilia B and Î²-thalassemia in Chinese patients (Liu et. al., 1998). In my opinion if a known wild-type (WT) sequence sample was also subjected to FF the sequencing step could be removed, as this could be used to compare a fingerprint of unknown and known sequence and to identify known mutations. This would be especially useful in the detection of previously unknown mutations particularly where only specific groups - whether ethnicity-; nationality-, environmentally-or geographically-based - possess them.

Multiplex Amplifiable Probe Hybridisation

Multiplex Amplifiable Probe Hybridisation (MAPH) was first described by Armour and colleagues in 2000 (Armour et. al., 2000). The general principle of MAPH (Fig 20) is the hybridisation of denatured test DNA with a set of amplifiable probes (with each probe specific to a unique region of the genome) on a nylon membrane, the unbound probes are washed off and the bound probes are recovered by stringent washing, amplified by PCR and separated by Polyacrylamide gel electrophoresis (PAGE) (Armour et. al., 2000; Sellner and Taylor, 2004). As only specific hybridisation occurs the amount of each probe retrieved is equal to the copy number of the corresponding test DNA sequence (Armour et. al., 2000). This technique could be used to detect copy number variants (CNVs) in disorders, as well as in evolutionary studies (Armour et. al., 2000). White S and colleagues improved upon MAPH in their study to scan for copy-number variants in Duchenne muscular dystrophy (DMD) patients, by labelling the PCR-products fluorescently and utilising a 96-capillary sequencer (ABI 3700) which can simultaneously analyse 96 samples in approximately 4 hours (White et. al., 2002).

Multiplex Ligation-dependent Probe Amplification

Multiplex ligation-dependent probe amplification (MLPA) was introduced in 2002 by Schouten and colleagues (Schouten et. al., 2002). Multiplex ligation-dependent probe amplification (Fig 21) utilises a similar principle to MAPH but differs in that oligonucleotide probes are amplified instead of sample DNA. Also MLPA does not require that sample nucleic acids are immobilised and excess probe need be removed (Sellner and Taylor, 2004). MLPA probes consist of two halves; one in which the target-specific sequence is flanked by a universal primer and the other which has the target-specific sequence and universal primer at opposite ends with a "stuffer sequence" (a random fragment of variable length) in between them. MLPA requires a thermocycler, electrophoresis equipment and a sequencer (Schouten et. al., 2002).

Figure 21: The MLPA technique. Extracted from Schouten et. al., 2002

A major disadvantage of MLPA when it was first developed its lengthy probe design process and probe preparation (Sellner and Taylor, 2004) has been compensated for as probe mixes can be purchased online at www.mlpa.com although probe mixes are only commercially available for specific diseases. Probe-specificity of MLPA is very high, avoiding non-specific binding (Schouten et. al., 2002). In addition it is highly sensitive and can be used to detect the gain or loss of a copy of a single exon as well as distinguish between two sequences which are different by as few as one nucleotide (SNP detection) (Schouten et. al., 2002). Gille and colleagues utilised MLPA to screen for genomic deletions of MSH2 and MLH1 [human mismatch repair (MMR) genes] in HNPCC, MLPA was found to be fast and efficient in testing for genomic deletions in MMR genes (Gille et. al., 2002). Since the screening of both genes could be done in a single reaction, MLPA can be adapted for use in many different applications by probe modification (Gille et. al., 2002). In a review comparing MAPH and MLPA Sellner and Taylor noted that MLPA was more advanced than MAPH, as MAPH had a higher risk of contamination as their probes were inheritantly amplifiable whereas ligation "activates" MLPA probes (Sellner and Taylor, 2004). Additionally MAPH has a washing step which could contaminate the samples (Sellner and Taylor, 2004). MLPA can also be automated making it higher throughput than MAPH (Sellner and Taylor, 2004).Eldering and colleagues modified the technique to be able to detect gene expression levels of gene transcripts over an extensive range - enabled by the use of a Reverse Transcription (RT) primer for each probe target sequence (Eldering et. al., 2003). The RT primer is designed by to be complementary to the downstream RNA sequence of the probe target. This modification of MLPA is known as RT-MLPA. Their study provided proof that RT-MLPA can be used in anti-inflammatory drug studies as well as studies investigating chemotherapy response in cancer patients (Eldering et. al., 2003). Methylation-specific MLPA (MS-MLPA) by Nygren and colleagues is another example of probe adaptation (Nygren et. al., 2005). It is used to detect changes in methylation patterns in addition to CNVs (Nygren et. al., 2005). Ligation is performed simultaneously with methylation sensitive endonuclease digestion, which digests DNA-probe complexes in which the CpG site is unmethylated resulting in no amplification product being formed. Methylation sensitive endonuclease does not digest DNA-probe complexes in which the CpG site is methylated. This technique can make use of paraffin-embedded tissues making it a powerful tool in tumour classification of tissue slices which were used in prior histological analysis. Bunyan DJ et. al. (2007) added point-mutation-specific (PMS)-MLPA probes to conventional MLPA to modify MLPA to detect point mutations. The PMS-MLPA probes hybridize to the forward DNA strand to prevent interference with the MLPA probes detecting CNVs - also reduces false positives.

Multiplex ligation-dependant genome amplification

Multiplex ligation-dependent genome amplification is based on the selector technique (Fig. 22) described by Dahl and colleagues in 2005 (Dahl et. al., 2005). Sample genomic DNA is subjected to restriction enzyme digestion to create distinct target nucleic acid end-sequences (Dahl et. al., 2005). The distinct target nucleic acid end-sequences hybridize to oligonucleotide constructs, known as selectors, which have target-complementary end-sequences (Dahl et. al., 2005). These target-complementary end-sequences are coupled by a general linking sequence (Dahl et. al., 2005). The selector is a ligation template which directs the target DNA to circularize (Dahl et. al., 2005). Background noise is reduced via endonuclease treatment to degrade linear DNA and subsequent multiplex amplification using a single universal PCR primer which recognises the general linkage sequence in the selectors (Dahl et. al., 2005).

Figure 22: The Selector technique. Extracted from Dahl et. al., 2005

Isaksson and colleagues based the Multiplex ligation-dependant genome amplification (MLGA) (Fig. 23) on this concept (Isaksson et. al., 2007). It was stated to be a more efficient mutation technique than MLPA, as genomic DNA was amplified instead of probe molecules and one probe is needed per target rather than two (Isaksson et. al., 2007). Probe manufacturing is easier and cheaper in MLGA as conventional oligonucleotide synthesis is utilised (Isaksson et. al., 2007). The total time for one experiment - including electrophoresis - is 5 hours whereas MPLA is approximately 24 hours (Isaksson et. al., 2007). More samples are able to be multiplexed in MLGA as PCR product length is determined by the genome DNA sequence instead of the length of synthetic probes resulting in longer PCR products than MLPA (Isaksson et. al., 2007).

Figure 23: The MLGA technique. Extracted from Isaksson et. al., 2007

A major disadvantage of MLGA is its dependence on genomic DNA sequences which can lead to a bias in the rate of amplification resulting from sequence diversity has been prevented by the introduction of more strict in silico probe design conditions (Isaksson et. al., 2007).

High Resolution Melt Analysis

Closed-tube high resolution melt (HRM) analysis (Gundry et. al., 2003) (Fig. 24) evolved from the continuous monitoring of PCR amplification using fluorescence detected by the Lightcycler (Wittwer CT, Herrmann MG et. al., 1997; Wittwer CT, Ririe KM et. al., 1997). This method can be used to distinguish between samples differing by as little as a single base and in comparative studies between homozygote and heterozygote samples (Gundry et. al., 2003).

The use of a 5'-fluorescently labelled primer allows strand disassociation (from dsDNA to ssDNA) to be monitored in real time as the temperature is increased (Gundry et. al., 2003). Using fluorescence strength a melting profile (a curve depicting the ssDNA-dsDNA transition) can be produced directly after PCR, as the fluorescent dyes are intercalating (binds in the grooves of the dsDNA) the fluorescent strength would decrease as the molecule transitions to ssDNA (Gundry et. al., 2003). Product reassociation is prevented by limiting the period the product is near its Tms, as well as by ensuring a fast heating rate and low concentration of Mg2+ (Gundry et. al., 2003).

Wittwer and colleagues compared the suitability of two fluorescent dyes, i.e. LCGreen and SYBR Green I, in mutation detection when 100ng/10ÂµL of DNA was used. Each dyes effect on PCR amplification was studied and it was concluded that LCGreen (Ëƒ90% content) could be used in higher saturation (the amount of dye that is allowed to bind to the DNA) than SYBR Green I (Wittwer et. al., 2003). LCGreen can be allowed to saturate more than 90% of the DNA whereas SYBR Green I had a complete inhibitory effect on amplification when the dye bound to more than 50% of the DNA (Wittwer et. al., 2003). The suitability of HRM for rapid screening of BCR-ABL KD mutations was proved by Poláková and colleagues in 2008 (Poláková KM et. al., 2008). Poláková and colleagues also proved that the LCGreen I does not interfere with the subsequent direct sequencing of positive samples, reducing the time required to do the entire assay (Poláková et. al., 2008). Ugo and colleagues recently concluded that HRM is a high-throughput detection method which can be utilised in routine diagnostic laboratory assays and is also reproducible inter-laboratory and inter-instrument in their study on Polycythemia Vera JAK2 Exon 12 mutations (Ugo et. al., 2010).

Conclusion

Newer techniques and those currently under construction are founded on principles validated by other older techniques. Mutation detection technique evolution is interdependent as a technique's evolution is influenced by financial, environmental factors and time-constraints but the development and improvement of other techniques allow this evolution to take place.

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